Advanced bioanalytical mass spectrometry methods for investigating protein covalent binding by reactive metabolites

药物代谢的主要作用是使机体从外源药物中解毒，使其更具极性，从而易于清除。然而，一些药物被生物活化成活性代谢物，具有与蛋白质共价结合的潜力。这种结合被认为是特异性药物反应（IDR）的重要原因。因此，反应性代谢物的形成代表了药物化合物的重大责任。这些活性代谢物可以在体内与蛋白质共价结合并引起显著的毒性，但其确切机制尚不清楚。我们研究项目的目标是设计新的方法来研究这些毒性相互作用。本研究的主要重点是设计新的策略，利用基于小分子和蛋白质组学的质谱工具的组合，进一步了解活性代谢物与蛋白质的共价结合，目的如下：***1)药物的代谢谱分析***2)评估蛋白质与特定蛋白质的结合***3)在体外鉴定蛋白质靶点。***4)进行体内实验，测量观察到的加合物的生物学相关性，并有可能发现反应性代谢物介导的毒性的特定生物标志物。***科学方法：将采用先进的HPLC-MS/MS分析，结合复杂的数据处理工具，对代谢物和修饰蛋白进行结构解析。基于我们之前的研究，我们的目标是用新的策略来研究反应性代谢物-蛋白质结合来扩展这项工作。这将涉及使用两种互补的方法从体外和体内模型中识别蛋白质靶点及其修饰位点。第一种方法涉及使用多维色谱耦合HR-MS/MS，第二种方法将使用我们研究小组开发的基于树脂的点击化学方法，使用选定的炔类药物进行修饰目标的选择性纯化。在可能的情况下，将比较两种方法的结果，以确定给定反应性代谢物的蛋白质靶标。一旦确定了蛋白质靶点，我们就可以在不同条件下（体外和体内）检查加合物的形成，以便更好地了解这些反应性代谢物与蛋白质共价结合的机制。为此，我们将开发新的方法来测量与感兴趣的选定蛋白质的结合程度。***影响：将为研究反应性药物代谢产物修饰蛋白质的机制设计新的策略。使用已知参与蛋白质共价结合的药物，将采取系统的方法来识别蛋白质靶点并阐明确切的结合位点。这项研究应该对更好地理解药物生物活化及其后果产生非常重要的影响，从而对人类健康产生直接影响。**

The main role of drug metabolism is to detoxify the body from xenobiotics, rendering them more polar and thus easily cleared. Some drugs, however, are bioactivated into reactive metabolites with the potential to covalently bind to proteins. This binding is considered to be an important cause of idiosyncratic drug reactions (IDR). As a result, the formation of reactive metabolites represents a significant liability for pharmaceutical compounds. These reactive metabolites can covalently bind to proteins in vivo and cause significant toxicity, however the exact mechanisms are still quite unclear. The goal of our research program is to design new methods to study these toxic interactions. The primary focus of this research is devising novel strategies to further understand reactive metabolite covalent binding to proteins using a combination of small molecule and proteomic-based mass spectrometry tools with the following objectives:***1) Metabolic profiling of drugs***2) Assess protein binding to specific proteins***3) Identify protein targets in vitro, and***4) Perform in vivo experiments to measure the biological relevance of adducts observed with the possibility of discovering specific biomarkers for reactive metabolite-mediated toxicity.***Scientific approach: Advanced HPLC-MS/MS analysis, combined with sophisticated data processing tools, will be employed for the structural elucidation of metabolites and modified proteins. Based on our previous research, we are aiming to expand on this work with new strategies for studying reactive metabolite-protein binding. This will involve identifying protein targets and their modification sites from both in vitro and in vivo models using two complementary approaches. The first involves using multidimensional chromatography coupled to HR-MS/MS and the second approach will use selected alkyne analogs of drugs for selective purification of modified targets using a resin-based click chemistry method developed in our research group. When possible, results from both methods will be compared for confirming protein targets of a given reactive metabolite. Once protein targets are identified, we can examine adduct formation under different conditions (in vitro and in vivo) in order to better understand the mechanism of protein covalent binding by these reactive metabolites. For this, we will develop novel methods for measuring the extent of binding to selected proteins of interest.***Impact: New strategies will be devised for studying the mechanisms of protein modifications by reactive drug metabolites. Using drugs known to be involved in protein covalent binding, a systematic approach will be taken to identify protein targets as well as elucidate the exact site of binding. This research should have very important consequences for better understanding drug bioactivation and its consequences, and therefore a direct impact on human health. **